Abstract
We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the β-subunit of human chorionic gonadotropin gene 6 and pre– trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant β-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.
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Acknowledgements
We thank Christopher D.J. Pearce and Bruce M. Cameron Jr. for enabling this project, Virginia G. Johnson (Food and Drug Administration) for providing DT-A clones, and Patrick M. O'Connor (National Cancer Institute) for useful discussions. Personal thanks (L.G.M.) to Carl R. Merril. This work was supported in full by Proteome Sciences plc.
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Puttaraju, M., Jamison, S., Mansfield, S. et al. Spliceosome-mediated RNA trans-splicing as a tool for gene therapy . Nat Biotechnol 17, 246–252 (1999). https://doi.org/10.1038/6986
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DOI: https://doi.org/10.1038/6986
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